Project Summary Inflammatory bowel disorders represent insufficient epithelial repair and colorectal cancer (CRC), the second leading cause of cancer deaths in the United States, reflects disruption of intestinal stem cell (ISCs) homeostasis. ISCs depend in part on underlying mesenchymal signaling gradients that balance proliferation versus differentiation capacity of crypt cells, including ISCs. Moreover, ISCs regenerate efficiently after ablation, by de-differentiation of crypt cells. It is unknown which signals or mesenchymal cells promote this regenerative process. My preliminary studies uncover three distinct mesenchymal cell types, two of which orchestrate BMP signaling gradients. High PDGFRA- expressing cells, which correspond to recently described telocytes, lie closest to the epithelium, are most abundant at the villus base, and express high levels of BMP ligands. Low PDGFRA-expressing cells contain two subpopulations: a CD81+ fraction found exclusively below crypts and expressing high levels of the BMP antagonist Gremlin1, and a CD81- fraction (CP cells) which resides above and around crypts but lacks Grem1. In co-cultures of isolated crypt epithelium with each of these strictly purified cell types, CD81+PDGFRAlo cells functionally replace all growth factor-supplemented media, while CP cells do so minimally and telocytes do not. Furthermore, ablation of Grem1+ cells in vivo results in loss of ISCs, hence I call these cells trophocytes. How each of these distinct mesenchymal cell populations regulates fetal ISC plasticity during development and ISC regeneration during loss, is unknown. I hypothesize that telocyte, CP, and trophocyte functions evolve during development, leading to genesis of a stable crypt- villus niche and that some of the same populations promote crypt regeneration after ISC ablation. In Aim 1, I will investigate the molecular profiles and functions of telocytes, CP cells, and trophocytes during mouse fetal ISC development. I will determine when these cells arise during development, investigate their full transcriptional profiles, and use an established crypt co-culture method to define their supportive roles. In Aim 2, I will test how these mesenchymal cells respond to ISC loss and promote crypt regeneration. Using in vivo assays of ISC regeneration, combined with bulk and single-cell RNAseq and in vivo genetic knockouts, I will test which cells promote ISC regeneration and specifically if this occurs through supraphysiologic BMP inhibition. Collectively, these studies will provide fundamental insights into the development and regulation of ISCs in fetal development and regeneration, with broad implications for intestinal biology and disease.